Produce Steviol Glycosides in Engineered Yeast

Steviol glycosides are natural products isolated from Stevia rebaudiana leaves, and are widely used as high intensity, low-calorie sweeteners.  Naturally occurring steviol glycosides have the same base structure (steviol) and differ in the content of carbohydrate residues (e.g. glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the diterpene steviol. Stevia extracts are commercially available, which typically contain stevioside (ST) and rebaudioside A (Reb A) as the primary compounds.  Although these two compounds are much sweeter than sucrose on per unit base, they tend to have strong off-flavor taste, limiting their applications in foods and beverages. Other steviol glycosides, which demonstrate many desirable taste properties, typically are present in Stevia extracts at much lower concentrations. 

The majority of the steviol glycosides are formed by several glycosylation reactions of steviol, which typically are catalyzed by the UDP-glycosyltransferases (UGTs) using uridine 5’-diphosphoglucose (UDP-glucose) as a donor of the sugar moiety.  In plants, UGTs are a very divergent group of enzymes that transfer a glucose residue from UDP-glucose to steviol.  In order to identify the key UGTs in the biosynthesis pathway of steviol glycosides, bioinformatics analysis was performed and selected UGT candidate genes were enzymatically characterized. This work identified several UGTs that demonstrated 1, 3-13-O-glucose, 1, 3-19-O-glucose, 1, 2-13-O-glucose, 1, 2-19-O-glucose and/or 1, 6-13-O-glucose glycosylation activity to produce 11 steviol glycosides including 6 known steviol glycosides (rebaudioside G, rebaudioside KA, rebaudioside A, rebaudioside E, rebaudioside D and rebaudioside M) and 5 novel steviol glycosides. Furthermore, we can produce these 11 steviol glycosides in large scale by using engineered UGT proteins and recombinant yeast cells. This recombinant yeast system has produced yields as high as 93% while providing a novel approach to successfully produce these desirable minor steviol glycosides at an industrially relevant scale.